The broad, long-term objectives of this proposal are to determine at a molecular level, the mechanisms of the Pol II general transcription factors and how their activity can be regulated. This study is important because understanding of the Pol II transcription machinery is essential to understanding how gene-specific regulatory factors function. This is because the transcription machinery is the ultimate target of gene- specific regulatory factors. Understanding the fundamental process of gene control will provide a basis for understanding many types of diseases such as cancer which often result from aberrant regulation of transcription initiation. In addition, some diseases involving defects in DNA repair are caused by mutations in the Pol II general factors. The specific aims of this project are to (i) study through molecular, genetic, and biochemical methods, the function of TFIIA, a regulatory general transcription factor. We will isolate suppressors of conditional TFIIA mutations, continue a structure-function analysis of TFIIA function, and characterize at least two inhibitors of Pol II transcription whose inhibitory activity is blocked by TFIIA. (ii) We will continue our purification of the Pol II general transcription machinery from yeast so we can use these factors in our study of general factor function. (iii) we will use yeast genetics to isolate and characterize activator independent mutations in TBP (the TATA binding protein) and/or TFIIB to study the mechanism of gene regulation. These mutations will allow us to test two specific models for how acidic transcriptional activators act. Our work will involve molecular techniques such as gene cloning, mutagenesis and PCR, biochemical fractionation, in vitro transcription, and protein chemistry, as well as yeast molecular genetics.